Baocheng Pan

Specific Radiation-Damage Can Be Used To Solve Macromolecular Crystal Structures

The use of third generation synchrotron sources has led to renewed concern about the effect of ionizing radiation on crystalline biological samples. In general, the problem is seen as one to be avoided. However, in this paper, it is shown that, far from being a hindrance to successful structure determination, radiation damage provides an opportunity for phasing macromolecular structures. This is successfully demonstrated for both a protein and an oligonucleotide, by way of which complete models were built automatically. The possibility that, through the exploitation of radiation damage, the phase problem could become less of a barrier to macromolecular crystal structure determination is discussed.

Crystal structure of an RNA purine-rich tetraplex containing adenine tetrads: Implications for specific binding in RNA tetraplexes

Purine-rich regions in DNA and RNA may contain both guanines and adenines, which have various biological functions. Here we report the crystal structure of an RNA purine-rich fragment containing both guanine and adenine at 1.4 A resolution. Adenines form an adenine tetrad in the N6-H em leader N7 conformation. Substitution of an adenine tetrad in the guanine tetraplex does not change the global conformation but introduces irregularity in both the hydrogen bonding interaction pattern in the groove and the metal ion binding pattern in the central cavity of the tetraplex. The irregularity in groove binding may be critical for specific binding in tetraplexes. The formation of G-U octads provides a mechanism for interaction in the groove. Ba(2+) ions prefer to bind guanine tetrads, and adenine tetrads can only be bound by Na(+) ions, illustrating the binding selectivity of metal ions for the tetraplex.

An eight-stranded helical fragment in RNA crystal structure: Implications for tetraplex interaction

Multistranded helical structures in nucleic acids play various functions in biological processes. Here we report the crystal structure of a hexamer, rU(BrdG)r(AGGU),at 1.5 A resolution containing a structural complex of an alternating antiparallel eight-stranded helical fragment that is sandwiched in two tetraplexes. The octaplex is formed by groove binding interaction and base tetrad intercalation between two tetraplexes. Two different forms of octaplexes have been proposed, which display different properties in interaction with proteins and nucleic acids. Adenines form a base tetrad in the novel N6-H em leader N3 conformation and further interact with uridines to form an adenine-uridine octad in the reverse Hoogsteen pairing scheme. The conformational flexibility of adenine tetrad indicates that it can optimize its conformation in different interactions.

Near-atomic resolution crystal structure of an A-DNA decamer d(CCCGATCGGG): cobalt hexammine interaction with A-DNA.

The structure of the DNA decamer d(CCCGATCGGG) has been determined at 1.25 A resolution. The decamer crystallized in the tetragonal space group P4(3)2(1)2, with unit-cell parameters a = b = 44.3, c = 24.8 A and one strand in the asymmetric unit. The structure was solved by the molecular-replacement method and refined to R(work) and R(free) values of 16.3 and 18.5%, respectively, for 5969 reflections. The decamer forms the A-form DNA duplex, with the abutting crystal packing typical of A-DNA. The crystal packing interactions seem to distort the local conformation: A5 adopts the trans/trans conformation for the torsion angles alpha and gamma instead of the usual gauche(-)/gauche(+) conformations, yielding G*(G.C) base triplets. The highly hydrated [Co(NH(3))(6)](3+) ion adopts a novel binding mode to the DNA duplex, binding directly to phosphate groups and connecting to N7 and O6 atoms of guanines by water bridges. Analysis of thermal parameters (B factors) shows that the nucleotides involved in abutting crystal packing are thermally more stable than other nucleotides in the duplex.

Structure of an RNA dodecamer containing a fragment from SRP domain IV of Escherichia coli

The crystal structure of an RNA dodecamer, r(GCGUCAGGUC(Br)CG)/r(CGGAAGCAG(Br)CGC), containing a fragment from the signal recognition particle (SRP) RNA (domain IV) of Escherichia coli, has been determined at 1.7 A resolution with 21 666 independent reflections and an R(work) and R(free) of 20.1 and 22.5%, respectively. The structure exhibits a novel crystal packing pattern for RNA oligomer duplexes: one end of the duplex adopts the stacking interaction, while the other end adopts the abutting interaction in the minor groove. The symmetric loop of the SRP, r(CAGG)/r(AGCA), in the center of the dodecamer forms two different conformations of the A.C mismatch, a sheared G.G and a symmetrical G.A mismatch. These four mismatches present a unique surface for the abutting interaction. The involvement of the two A.C mismatches in the abutting interaction implies that these mismatches are the important sites for interaction with proteins. The conformation of the symmetric loop is greatly stabilized by hydrated metal ions, which display flexibility in adjusting their geometry and coordination in interaction with nucleic acids. Comparison with other crystal structures of fragments of 4.5S RNA indicates that the conformation of the symmetric loop is independent of the asymmetrical loop in domain IV.

Synthesis, Purification and Crystallization of Guanine-rich RNA Oligonucleotides

Guanine-rich RNA oligonucleotides display many novel structural motifs in recent crystal structures. Here we describe the procedures of the chemical synthesis and the purification of such RNA molecules that are suitable for X-ray crystallographic studies. Modifications of the previous purification methods allow us to obtain better yields in shorter time. We also provide 24 screening conditions that are very effective in crystallization of the guanine-rich RNA oligonucleotides. Optimal crystallization conditions are usually achieved by adjustment of the concentration of the metal ions and pH of the buffer. Crystals obtained by this method usually diffract to high resolution.

Structure of d(ITITACAC) complexed with distamycin at 1.6 Å resolution

The crystal structure of the DNA octamer d(ITITACAC)(2) complexed with distamycin has been determined at 1.6 A resolution and refined to a final R(work) and R(free) of 17.0 and 20.7%, respectively. Two molecules of distamycin bind to the DNA duplex in an antiparallel side-by-side fashion. Each drug molecule covers five base pairs of the DNA duplex, with its amide groups hydrogen-bonding to bases in the proximal DNA strand. These two antiparallel drug molecules are stacked together with the pyrrole rings of one molecule stacking against the amide groups of the other. The present structure emphasizes the features of alternating DNA octamers in interaction with distamycin.